Studies On Ciliature Microtubular Organelles And Tubulins In The Hypotrich Ciliate Urostyla Grandis

The life activities in single-cell protozoan are equivalent to the exercise of all the physiological function in multicellular, and any cells in multi-cellular organisms can not compare with them in the complexity of cell structure.Hypotrichous ciliates belong to the groups with the most complex cell structure and the highest evolution in the protozoa. The cells contain a variety of structural components forming the outer layer--the cortex, especially the developed cytoskeleton components within which cause the concern of researchers. Therefore, to study the morphology and the regulation in cell morphogenesis of cortical cytoskeleton become a hot field of cell biology. Microtubules are the main component of cortical cytoskeleton in hypotrichous ciliates,in the two microtubule systems of cortical microtubules and non-ciliary microtubules, microtubules form the oral-ciliature microtubules,soma-ciliature microtubules and its associated microtubules which are the most complex microtubule system in the Ciliates. Using hypotrichous ciliates as the material to study the construction characteristics of microtubular organelles and the differentiation, assembly and position of microtubule components during morphogenetic,can not only provide basic data for the study of the evolutionary relationship between ciliate species but also have important scientific significance in the exploration on the regulation of the structure pattern formation in eukaryotic cells in-depth, Including providing the scientific basis during the gene regulation and non-gene regulation process, and making further revelation of regulation mechanism in different cellular levels.In this aticle, we use Urostyla grandis as materials to detect its cilia-structure differentiation in the term of morphology and morphogenesis through the method of Flutax direct fluoreseent-labeled and anti-αtubulin antibody.we found that the cortical microtubule skeleton is different in the different lifecycle. This revealed the law of its morphogenesis tubulin differentiation and positioning.And we also use SDS-PAGE to analyze its protein components.The results are as follows:1.Ciliature microtubules and ciliature base-associated microtubules of Urostyla grandisThe ciliature microtubular organelles in the ventral cortex of Urostyla grandis were visualized by direct fluorescent labeling of FLUTAX and the anti-a tublin antibody.The organelles consist of adoral zone of membranelles(AZM),undulating membranes(UM),frontal-midventral-transverse cirri(FVTC),left and right marginal cirri (L-and RMC)and the base-associated microtubules,etc.These base-associated microtubules is comprise of membranlle brackets of AZM,its associated oral rib-wall microtubules, base body brackets of UM, anterior longitudinal microtubules (ALM)and posterior longitudinal microtubules (PLM) of frontal cirri(FC),ALM of TC,ALM,PLM and transverse microtubules(TM) of midventral cirri(VC)and L-and RMC. According to the results, despite ciliature base-associated microtubules of hypotrichous ciliates have a basic structural component,the composition, differentiation characteristics, location and orientation, level of development are different.Therefore,the results provide new evidence for the morphology of cortex ciliated cells and diversity of microtubules construction.2.Morphogenesis of microtubular organelle in Urostyla grandis during Period of vegetative division and defferetiationDuring the process of the morphogenesis of Urostyla grandis in the ventral cortex.AZM of proter was generated from the renewed lapel Part of AZM(AZM-L), the old AZM-C as well as the conserved minor intersection between AZM-L and AZM-C.During the morphogenesis and differentiation of the ciliatures,the old frontal cirri,transverse cirri and marginal cirri in the non-primordium-field could persist and remain unchanged for a long time and they might play a role in the movement of ciliates during the developing period of the new ciliatures. The base-associated microtubules in the ventral cortex of Urostyla grandis possess the basic characteristics of hypotrich ciliates,it also has some special mode of composition and occurrence.The microtubular organelles of old ciliatures might play an important role in the location and substance supply of new structures when these structures begain to occur and develop. During the period of defferetiationand maturation of the new structures,the old ciliatures seemed to play a similar role when they gradually disintegrated and disappeared.3.Analysis on the protein components of Urostyla grandisThe protein components of Urostyla grandis were investigated using method of biochemical extraction, SDS-PAGE and anti-α, anti-γtubulin immunoblotting. The results are as follows:all protein components of Urostyla grandis were expressed between 14kDa to 66kDa. There are darker strips near 20kDa and 26kDa-66kDa.In addition to confirm the molecular weights ofα-andγ-,to be 50 kD,48 kDa. It proved thatα-andγ-tubulin microtubules in the ciliate cytoskeleton are conservative.